Defense Date


Document Type


Degree Name

Doctor of Philosophy


Pharmacology & Toxicology

First Advisor

Ronald P. Rubin


The present report describes the characteristics and regulatory properties of phosphatidylinositol (Ptdlns) 4-kinase activity in rat exocrine pancreas. The membrane associated Ptdlns 4-kinase displayed a broad pH profile with optimal activity at neutral to alkaline pH. Carbachol (CCh) elicits a concentration- and time-dependent increase in Ptdlns 4-kinase activity in homogenates derived from agonist-stimulated acini. This effect was blocked by N-methylscopolamine and mimicked by muscarine. The enzyme had an apparent Km for Ptdlns and ATP of 4 and 60 uM, respectively. CCh caused no discernible change in the Km for either Ptdlns or ATP, but did produce a modest increase in the Vmax. The cell permeable diacylglycerol analogues dioctanoylglycerol (DiC8) and oleoyl acetylglycerol (OAG) also produced a concentration-dependent increase in Ptdins 4-kinase activity which was blocked by the protein kinase c inhibitor, staurosporine. Cell permeable monobutyryl cAMP caused an increase in PtdIns 4-kinase activity when added to intact acini. This agent caused a significant decrease in the apparent Km for ATP but had no effect on PtdIns utilization or the Vmax of the reaction. Moreover, pretreatment of the acinar homogenate with the catalytic subunit of protein kinase A (PKA) produced a concentration-dependent increase in enzyme activity suggesting that phosphorylation may be involved in the regulation of Ptdins 4-kinase. Epidermal growth factor (EGF) elicited a concentration-dependent increase in PtdIns 4-kinase activity in homogenates derived from agonist-stimulated pancreatic acini. The combination of CCh and EGF produced a response which was not synergistic or additive. EGF, unlike CCh, failed to cause [32^P]Ptdins(4,5)P2 breakdown, suggesting different mechanisms for the increase in Ptdins 4-kinase activity induced by EGF and CCh. Furthermore, exposure of pancreatic acinar cells to EGF failed to elevate cyclic AMP levels, suggesting that a third pathway exists for the regulation of Ptdins 4-kinase activity in the exocrine pancreas. We conclude that PtdIns 4-kinase represents a key component of the signaling pathways utilized by various receptor agonists and that phosphoinositide synthesis is under direct and tight regulation by PKC- and PKA-dependent pathways.


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